1. Field of the Invention
This invention relates to an assay method and reagent composition for determining chloride levels in fluids, and more particularly to a direct colorimetric method which avoids a requirement of removing protein from the samples.
2. Importance of Accurate and Reliable Chloride Measurements
Chloride is the major extracellular anion in human body fluids, and it is thus significantly important in maintaining proper water distribution, osmotic pressure, and normal electrolyte (anion-cation) balance in the human body. Low serum chloride values may be found with prolonged vomiting, extensive burns, metabolic acidosis, Addisonian crisis, and renal diseases. Elevated chloride values, on the other hand, are associated with dehydration, congestive heart failure, hyperventilation and urinary obstructions. Determination of chloride in sweat is useful in the diagnosis of cystic fibrosis, where the secretion of chloride in sweat may be up to five times as high as that of healthy individuals.
Because of the vitally important roles chloride ions play in the normal functioning of life processes, and in view of the effects of diseases and other body malfunctions on the amount of chloride ions in the body, it has long been recognized that it is necessary to be able to accurately and reliably determine or measure chloride levels in serum and other body fluids in order to aid the physician in proper diagnosis and treatment. In addition, it is correspondingly often necessary that such measurements are able to be made quickly in response to an urgent or emergency request from a physician. Moreover, the ever-increasing recognition by clinicians of the need for frequent chloride determinations requires that the assay method desirably be suitable for multiple-purpose automated laboratory equipment.
3. Description of the Prior Art
A variety of direct and indirect methods for measurement of chloride has been reported in the literature. These methods may be described as mercurimetric titration, coulometric-amperometric titration, ion-specific electrode techniques, and colorimetric methods. Although these methods represent the prior art through the years, each has had certain disadvantages. For example, titration procedures tend to be time-consuming, and they are especially burdensome when the sample must be subjected to deproteinization procedures or other pre-treatment steps. Coulometric and ion-specific electrode techniques require the use of specialized laboratory instrumentation and equipment of limited utility. Further, these methods do not readily lend themselves to automation. Colorimetric methods, however, do not require the use of such highly specialized equipment or instrumentation and, in general, are adaptable to automation.
The most widely used of the automated colorimetric methods is the mercuric thiocyanate method. Unfortunately, however, this method suffers from several drawbacks. First, the method is not linear, and thus multiple standards and cumbersome calibration curves must be used to obtain valid results. Non-linearity is a further problem when assaying chloride in urine samples, which often have chloride levels lower than the lowest calibrator used to prepare the curve. Moreover, the method uses high concentrations of hazardous mercury which must be disposed of as waste. Also, the thiocyanate has a greater affinity for bromide and iodide than for chloride, thereby causing serious interference when these ions are present in an assay for chloride specifically. Finally, most mercuric thiocyanate methods involve protein removal, usually by dialysis, which can introduce further errors into the assay.
In what might be considered to be the closest of the prior art, Fried et al. (J. Clin. Chem. Clin. Biochem. 10, 1972, p. 280) describe a colorimetric method for the direct determination of chloride using mercuric ions and the indicator 2,4,6-tripyridyl-s-triazine (TPTZ). Feldkamp et al. (J. Clin. Chem. Clin. Biochem. 12, 1974, pp. 146-150) describe the use of a colorimetric mercuric TPTZ method in studying interfering halides on continuous flow analyzers. De Jong et al. (Clin. Chem. 26, 1980, pp. 1233 and 1234) describe a modification of the Feldkamp method which is also utilized with continuous flow analyzers. The de Jong modification was reportedly made to overcome the shortcomings of the Feldkamp procedure. As stated on page 1233 of the de Jong publication, "The automated method of Feldkamp et al. . . . involving mercuric 2,4,6-tripyridyl-s-triazine (TPTZ) as proposed by Fried et al. . . . is excessively noisy owing to the very high dilution required by this sensitive reagent, is critically dependent on mercuric nitrate flow, and does not decrease the amount of mercury used. The method, however, is linear and neither iodide nor bromide react preferentially." In spite of the achievements of de Jong, the Feldkamp method described still suffers from several drawbacks, as now specified.
First, the method suffers from interference due to the presence of protein in the sample. Because of the very high sample dilutions used by these authors, the interference has presumably been minimized, but dialysis to separate the protein from the reaction is used by both Feldkamp and de Jong. Feldkamp tried to further avoid the protein problem by adding a fixed amount of albumin to the samples used in his study so that the effect from the protein would be identical in all samples tested. Fried did apply the mercuric TPTZ reaction to protein-containing samples, but he makes no mention regarding interference due to protein. Because of the high dilution ratios Fried used (1:860 and 1:1070), the protein effect is presumably minimized to at least some extent by diluting the protein out. With regard to the Fried publication, it should be pointed out that Fried discloses only the presence and concentration of two ingredients in the reagent he used, namely mercuric-TPTZ and ferrous sulfate. This limited disclosure would hardly enable the most highly skilled in the art to perform the assay described in the publication.
Furthermore, the reagent described by de Jong is reported to be stable for at least one month at room temperature. This would therefore require reagent preparation by the analyst each month, and would preclude the time-saving and convenience of obtaining the reagent in a commercially manufactured, shelf stable form.
Finally, because of the method's extreme sensitivity, samples to be assayed must either be diluted manually prior to performing the assay, or they must be diluted out as described by de Jong, that is, using two dialyzers in series, or by some other manipulative means.